Epigenetic changes, including DNA methylation, are a common finding in human malignancies. Research in[unreadable] chronic lymphocytic leukemia (CLL) has focused on genetic deletions that promote impaired apoptosis, but to[unreadable] date very few genes have been identified. In preliminary studies we have utilized Restriction Landmark[unreadable] Genomic Scanning (RLGS), a two-dimensional gel electrophoresis that allows detection of altered DNA[unreadable] methylation patterns, to study methylation in ten CLL patient samples. Our results strongly indicate that DNA[unreadable] methylation, via gene silencing, contributes significantly to the pathogenesis of CLL. Specifically, we[unreadable] demonstrated marked variation in the amount of aberrant methylation in patient samples ranging from 2.5-8.1%[unreadable] as compared to normal B cells. To fully establish the role of methylation in CLL and to exploit these alterations[unreadable] in the clinic, we propose more detailed studies outlined below. Our hypothesis for this Project is that DNA[unreadable] methylation contributes significantly to the development and progression of CLL. In Specific Aim 1, RLGS will[unreadable] be used on 100 CLL patients of different genotypes. RLGS profiles of CLL patients samples will be compared[unreadable] with CD19+ selected normal B-lymphocytes to identify novel CLL methylation sequences specific to CLL. In[unreadable] addition, methylation patterns between favorable and un-favorable genotypes will be compared to identify the[unreadable] most promising gene targets to pursue for investigation in Specific Aim 2. In Specific Aim 2, we plan to clone as[unreadable] many as 200 novel methylated sequences identified from aim 1 using genomic libraries that will guide the[unreadable] search for new tumor suppressor genes in CLL. Furthermore we propose experiments to perform a[unreadable] biostatistical evaluation of DNA methylation patterns in CLL. In aim 3 these genes will then be analyzed in a[unreadable] large cohort of CLL patient samples obtained from the national cooperative oncology groups, and results will be[unreadable] correlated with response to therapy, progression-free survival and overall survival. In Specific Aim 4, novel[unreadable] candidate cancer genes, DAPK1, ID4 and DERMO1, whose expression is altered by DNA methylation, will be[unreadable] studied in more detail in order to determine potential importance to the pathogenesis and progression of CLL.